Chromatin assembly and structure is highly regulated during DNA replication, gene expression, and progression through the cell cycle. Dysfunctions of epigenetic factors, including methylation states, are associated with a variety of cancers. Histone methyltransferases (HMTs), including lysine and arginine methyltransferases, are considered a new and important class of drug targets, however high throughput screening (HTS) assay formats are not available and reference inhibitors remain unknown. Establishment of HTS assays and reference compounds would have significant potential to help elucidating the function of these enzymes and to facilitate the development of anticancer agents. Reaction Biology Corporation (RBC) has developed extremely low cost reaction systems for many enzyme classes to serve markets for HTS drug discovery, large scale IC50 determinations and selectivity/toxicity profiling. RBC's HotSpot platform employs a gold standard for ultralow volume radioisotope assays for kinase profiling. This service product for kinase profiling is widely accepted in the drug discovery community for kinase inhibitor development. Based on these skills, RBC is developing a new assay platform using gold standard radioisotope assays for the large family of histone methyltransferases (HMTs). During Phase I, RBC has successfully developed over 13 methyltransferases by using our low cost radioisotope based format. These assays have been validated internally and by customers. We have conducted a trial HTS by using one of the enzymes and identified a few pan-methyltransferase inhibitors that are under further evaluation in both large panel profiling and in cell based assays. Eventually, these compounds could be used as research tools for epigenetic research, which is lacking reference compounds. In Phase II, we propose to expand this technology to add an additional 20 methyltransferases (Aim 1). In Aim 2, HTS campaigns using a 45,000 compound library of diverse structures against the RBC panel of methyltransferase will establish a database to drive structure-activity relationship (SAR) models. In Aim 3, all the individual hits will be profiled for in vitro selectivity and potency, candidates with good activity and clean structures will be further evaluated in cell based assays for methyltransferase inhibition, and lead compounds will go through further SAR studies. Through Phase II funding, RBC will be able to provide HTS and profiling service to cover majority of the human methyltransferases and providing tool compound for research activities. By the end of this funding, RBC will be the major driving force for providing most of the tools needed for epigenetic drug discoveries. After Phase II, RBC will looking for potential collaborations to further SAR studies on the inhibitors that we have discovered in the Phase II screening process. As a result of this funding, drug discovery labs will have access to a new class of industrial grade screening and profiling assays that do not exist today, and protein production in this area will be upgraded as well. PUBLIC HEALTH RELEVANCE: Chromatin assembly and structure is highly regulated during DNA replication, gene expression, and progression through the cell cycle. Dysfunctions of epigenetic factors, including methylation states, are associated with a variety of cancers. Histone methyltransferases (HMTs), including lysine and arginine methyltransferases, are considered a new and important class of drug targets, however high throughput screening (HTS) assay formats are not available and reference inhibitors remain unknown. Establishment of HTS assays and reference compounds would have significant potential to help elucidating the function of these enzymes and to facilitate the development of anticancer agents. Reaction Biology Corporation (RBC) has developed extremely low cost reaction systems for many enzyme classes to serve markets for HTS drug discovery, large scale IC50 determinations and selectivity/toxicity profiling. The HotSpot platform employs a gold standard for ultralow volume radioisotope assays for kinase profiling. This service product for kinase profiling is widely accepted in the drug discovery community for kinase inhibitor development. Based on these skills, RBC seeks to develop a new assay platform using gold standard radioisotope assays for the large family of histone methyltransferases.